Complete Genome Sequence of Aggregatibacter actinomycetemcomitans Strain IDH781.

We report here the complete genomic sequence and methylome of Aggregatibacter actinomycetemcomitans strain IDH781. This rough strain is used extensively as a model organism to characterize localized aggressive periodontitis pathogenesis, the basic biology and oral cavity colonization of A. actinomycetemcomitans, and its interactions with other members of the oral microbiome.

GENOME ANNOUNCEMENT

Aggregatibacter actinomycetemcomitans is a Gram-negative nonmotile facultative anaerobe of the oral microbiota implicated in the development of localized aggressive periodontitis (LAP) (1, 2). Despite the lack of a complete genomic sequence, IDH781, a rough strain, is commonly used as a model for pathogenesis and basic bacteriology studies (3–11). Rough strains of A. actinomycetemcomitans display the classic star-shaped colony morphology observed in clinical isolates from LAP patients, so they are more appropriate for the study of A. actinomycetemcomitans biology than those displaying a smooth colony phenotype (12, 13).

IDH781 was grown under anaerobic conditions (10% hydrogen, 10% carbon dioxide, and 80% nitrogen) on brain heart infusion agar supplemented with glucose, sodium bicarbonate, and yeast extract. The rough phenotype was confirmed microscopically. Cells were scraped from agar plates and lysed in 625 µg/ml proteinase K, 1.25 mg/ml lysozyme, and 2% sodium dodecyl sulfate. Genomic DNA was purified by phenol-chloroform/isoamyl extraction (14). DNA integrity was verified by agarose gel electrophoresis, purity was evaluated spectrophotometrically, and concentration was determined fluorometrically.

Sequencing libraries were constructed using the Pacific Biosciences 20-kb template preparation protocol and Sage Science’s BluePippin size-selection system with a 5-kb fragment size cutoff. Pacific Biosciences single-molecule real-time (SMRT) sequencing was performed on an RSII instrument using a single SMRT cell with P6-C4 chemistry and a 3-h movie, producing 7,601 polymerase reads (N50, 11,624 nucleotides [nt]) and 153,117 postfiltered subreads (N50, 6,741 nt). De novo assembly with the HGAP assembler (version 2.3) yielded a single contig supported by a mean coverage of 283-fold (15).

The genome was circularized by permutation to start at the dnaA gene and remove terminal duplications using Circlator (version 1.0.2), followed by resequencing using RS_Modification_and_Motif_Analysis (version 2.3) to correct errors at the original contig break and to detect DNA methylation motifs based on significant interpulse duration signals (15, 16). Two well-supported m6A motifs were detected, GATC and AGGAG (bold indicates the methylated residue), with >98% of motifs in the genome detected. Seven other unique nonpalindromic modifications were detected at low frequency; these consisted of 2 putative m6A motifs (34% and 30%), 1 putative m4C motif (11%), and 4 unknown base modifications (20%, 15%, 8%, and 4%). The significance and importance of these low-frequency interpulse duration signals remain unknown.

The final assembly was 2,291,252 bp, with a G+C content of 44.3%, consistent with other completed A. actinomycetemcomitans genomes. To verify strain identity and detect possible horizontal gene transfer events, genomic intervals were taxonomically assigned using Taxator-tk (version 1.3.1e) with the nonredundant-microbial_20140513 database (refpack from http://research.bifo.helmholtz-hzi.de/software) (17). All classified regions (39.8% of the genome) were assigned to the species A. actinomycetemcomitans.

Annotation was performed by NCBI using the Prokaryotic Genome Automated Annotation Pipeline (PGAAP, best-placed reference protein; GeneMarkS+; version 3.3) (18). The chromosome contains 2,206 genes, with 2,129 coding sequences, 19 rRNAs, and 54 tRNAs for all 20 amino acids plus selenocysteine, 4 noncoding RNAs (ncRNAs), and 3 predicted clustered regularly interspaced short palindromic repeats (CRISPRs).

Accession number(s).

The complete genome sequence of A. actinomycetemcomitans strain IDH781 has been deposited in GenBank under the accession number CP016553. The version described here is the first version.